Over 14,000 women in the U.S., each year die from epithelial ovarian carcinomas (1). Nearly 75% of patients present with advanced disease since, as of yet, no reliable means for early detection is available. The effective treatment of advanced or recurrent ovarian cancer is often compromised by both the intrinsic and acquired resistance of many ovarian carcinomas to one or more of the chemotherapeutic agents commonly employed in the treatment of this disease (2, 3). Without a practical means of predicting clinical response to therapeutic agents, treatment of any given patient is empirically based on the most successful agent or agents in a recent clinical trial rather than any objective assessment of individual tumor response. This is the functional equivalent of treating patients with septicemia with the combination of antibiotics found most effective in the treatment of such infections, without blood cultures and antibiotic testing of the cultured pathogens. While such an approach might be effective for a majority of patients many would die for lack of the appropriate antibiotic, which is the current situation for ovarian cancer and for that matter nearly all other solid tumors of adults.
To make treatment more effective, a number of assays have been proposed to help predict tumor cell responses to chemotherapeutic agents prior to treatment (2, 4-12). Various types of clonogenic assay have been used for many years to test cytotoxic compounds in vitro. Such assays are reasonably predictive of drug action in vivo and remain the standard against which other assays are compared but the best of them are not amenable to clinical situations given the low clonogenic efficiencies of most primary tumor samples in culture. Limited clinical success has been achieved in drug resistance studies performed on tumor tissue samples where a change in a metabolic parameter is measured after drug treatment as a surrogate for measurement of clonogenic survival (2, 4, 8, 13, 14). Assays for chemo-sensitivity such as ATP-TCA have shown promise in guiding the selection of suitable therapies on an individual basis (2, 9, 11, 15). Although not yet proven to be beneficial in prospective studies, results to date suggest that assays of surrogate markers of cell death could be effective tools to help guide the treatment of patients if the in vitro endpoint is actually a reasonable predictor of the in vivo response of the tumor from which the sample was obtained (16).
Resistance to programmed cell death (apoptosis) has been described as the “hallmark” of cancer transformation. The final effect of successful chemotherapy represents the consequences of the activation of the apoptotic machinery by the agents used: damage to DNA, microtubules and other cell components. Initially, the development of chemotherapeutic agents was based on the observation that tumor cells proliferate faster than normal cells which led to the original strategy of trying to, selectively if possible, block tumor cell DNA replication or cellular metabolism. At that time drug resistance was thought to arise from molecular changes inhibiting the drug/target interaction.
Caspases are highly specific proteases synthesized as zymogens and activated by cleavage which generates large and small subunits of the mature enzyme. Caspases can collaborate in the proteolytic cascade by activating themselves and each other (24, 25). Within these cascades, caspases can be divided into “initiator” caspases and downstream “effectors” of apoptosis. Initiator caspases, such as caspase-8 and 9, mediate their oligomerization and autoactivation in response to specific upstream signals. The effector caspases include caspases-3 and 7, which cleave cellular substrates and precipitate apoptotic death. (See, Human Caspase 3 nucleic acid (GenBank Accession NM—004346)41; Human Caspase 3 protein (GenBank Accession NP—004337)42; Human Caspase-7 nucleic acid (Caspase 7 nucleic acid, (GenBank Accession NM—0012227 (alpha), NM—033340 (beta), NM—033339 (gamma), NM—033338 (delta)); Human Caspase 7 protein (GenBank Accession NP—203125 (alpha), NP—203126 (beta), NP—203124 (delta))43.
The elucidation of the detailed molecular mechanism of apoptosis and its function in normal physiology has resulted in a better understanding the effect of chemotherapy and the mechanisms of resistance. It is now well documented that the induction of apoptosis in target cells is a key mechanism for most anti-tumor therapies, including chemotherapy, radiation, immunotherapy and cytokines (17-22). More recently, studies have applied measurement of the apoptotic response to the determination of chemo-sensitivity (18). These studies indicate that drug-induced apoptosis but not antiproliferative measurement, can predict tumor response to chemotherapeutic drugs (23). Furthermore, the in vitro response of tumor cells exposed to physiological doses of chemotherapeutic agents can be tested for sensitivity or resistance by employing markers of apoptosis which correlate with tumor cell death.
Practical assays to predict the likelihood of individual tumor sensitivity and which optimize the potential for efficient treatment and avoid the toxicities of ineffective drugs are needed to facilitate the choice of adequate treatment.